Middle-aged rats orally supplemented with gel-encapsulated catechin favorably increases blood cytosolic NADPH levels.

Green tea catechins are primarily known to function as free radical scavengers and have several beneficial uses. Orally supplemented catechin (OSC) was previously shown to increase mitochondrial heme and catalase levels in rat heart blood, however, its effect in the cytosol has not been elucidated. Here, we determined the effects of OSC in the rat heart blood cytosol. We used middle-aged (40 week-old) and young (4 week-old) rats throughout the study. We isolated blood cytosol, verified its purity, and determined heme, hydrogen peroxide (H2O2) levels, catalase (CAT) activities, gp91(phox) amounts, NADP and NAD pools, sirtuin 1 (SIRT1) and glutathione reductase (GR) activities, and free fatty acids (FFA). We established that OSC is associated with decreased heme-dependent H2O2 amounts while increasing heme-independent CAT activity. Moreover, we found that OSC-related decrease in NAD(+) amounts among middle-aged rats is associated to increased NADPH levels and SIRT1 activity. In contrast, we associated OSC-related decrease in NAD(+) amounts among young rats to decreased NADPH levels and increased SIRT1 activity. This highlights a major difference between catechin-treated middle-aged and young rats. Furthermore, we observed that cytosolic FFA and GR levels were significantly increased only among OSC-treated middle-aged rats which we hypothesize are related to increased NADPH levels. This insinuates that OSC treatment allows higher catechin amounts to enter the bloodstream of middle-aged rats. We propose that this would favorably increase NADPH amounts and lead to the simultaneous decrease in NADPH-related pro-oxidant activity and increase in NADPH-related biomolecules and anti-oxidant activities.

The assessment of the energy metabolism in patients with chronic fatigue syndrome by serum fluorescence emission.

CONTEXT: Chronic fatigue syndrome (CFS) is a debilitating fatigue illness that has unknown etiology and lacks an objective diagnostic marker. OBJECTIVE: To examine the metabolic component of CFS to determine if practitioners can use serum NAD(P)H concentration measurements to monitor metabolism and fatigue status in patients with CFS. DESIGN: The research team conducted a case-control study, comparing a group of patients who were diagnosed with CFS with a control group of healthy subjects. The team obtained venous blood samples from fasting patients to examine the serum NAD(P)H concentrations. SETTING: The study occurred at the Riordan Clinic in Witchita, Kansas. PARTICIPANTS: The study included 44 CFS patients at the Riordan Clinic and 30 healthy control participants. The CFS patients presented a spectrum of symptoms that had existed for at least 6 months: new, unexplained, persistent, or relapsing chronic fatigue that bed rest did not resolve and that was severe enough to reduce daily activity significantly by 50% in conjunction with headache, muscle pain, pain in multiple joints, and unrefreshing sleep. In the control group, the research team enrolled subjects without diagnosis of disease or injury. OUTCOME MEASURES: The research team determined levels of serum reduced nicotinamide adenine dinucleotides (NADH and NAD[P]H) by measuring serum fluorescence emission at 450 nm. The team then conducted sensitivity and specificity analyses. Results NAD(P)H concentrations in serum of CFS participants averaged 8.0 ± 1.4 (standard deviation [SD]) nmol/mL, while those in the healthy controls averaged 10.8 ± 0.8 (SD) nmol/mL, a statistically significant difference. Using a cut-off concentration of 9.5 nmol/mL, the research team attained a sensitivity of 0.73 and a specificity of 1.0. An analysis of receiver-operator characteristics yielded an area under the curve of 0.9. The research team compared serum NAD(P)H to several endocrine and metabolic lab parameters. Serum NAD(P)H was directly correlated with serum CoQ10 levels and inversely correlated with urine hydroxyhemopyrrolin-2-one levels. CONCLUSIONS: Based on these findings, the research team proposed using serum NAD(P)H, measured as an intrinsic serum-fluorescence emission, to monitor metabolism and fatigue status in patients with CFS. Following patients NAD(P)H levels over time may aid in selecting therapeutic strategies and monitoring treatment outcomes.

Efficacy of turmeric on blood sugar and polyol pathway in diabetic albino rats.

In the traditional system of medicine, Ayurveda, several spices and herbs are thought to possess medicinal properties. Among the spices, turmeric rhizomes (Curcuma longa. Linn.) are used as flavoring and coloring agents in the Indian diet everyday. In this research, we studied the effect of turmeric and its active principle, curcumin, on diabetes mellitus in a rat model. Alloxan was used to induce diabetes. Administration of turmeric or curcumin to diabetic rats reduced the blood sugar, Hb and glycosylated hemoglobin levels significantly. Turmeric and curcumin supplementation also reduced the oxidative stress encountered by the diabetic rats. This was demonstrated by the lower levels of TBARS (thiobarbituric acid reactive substances), which may have been due to the decreased influx of glucose into the polyol pathway leading to an increased NADPH/NADP ratio and elevated activity of the potent antioxdiant enzyme GPx. Moreover, the activity of SDH (sorbitol dehydrogenase), which catalyzes the conversion of sorbitol to fructose, was lowered significantly on treatment with turmeric or curcumin. These results also appeared to reveal that curcumin was more effective in attenuating diabetes mellitus related changes than turmeric.

Reduction of hydroxamic acids to the corresponding amides catalyzed by rabbit blood.

1. The hydroxamic acids N-hydroxyphenacetin and N-hydroxy-2-acetylaminofluorene were reduced to the corresponding amides, phenacetin and 2-acetylaminofluorene respectively by rabbit blood supplemented with both NAD(P)H and FAD. These reducing activities were found in erythrocytes but not in plasma, and were sensitive to inhibition by carbon monoxide and oxygen. When blood or erythrocytes were boiled, these activities were not abolished. 2. Haemoproteins such as haemoglobin and catalase exhibited the reductase activity in the presence of both NAD(P)H and FAD under anaerobic conditions. The activity was not abolished when the haemoproteins were boiled. 3. Haematin showed a significant reducing activity in the presence of these cofactors. The activity of haematin was also observed with the photochemically reduced form of FAD. 4. The reduction system in blood was composed of NAD(P)H, FAD and haemoglobin. Reduction appears to proceed in two steps, i.e. the reduction of FAD by NADH or NADPH, followed by the non-enzymatic reduction of the hydroxamic acids to the amides by reduced FAD, catalyzed by the haem group of haemoglobin in rabbit erythrocytes.

Optimal and stable conditions for the determination of erythrocyte glutathione reductase activation coefficient to evaluate riboflavin status.

To measure the activation coefficient (AC) of erythrocyte glutathione reductase (GR), suspended whole blood was lysed in a preincubation solution, with or without 100 microM flavin adenine dinucleotide (FAD). Upon addition of the reaction mixture, FAD concentration decreased about 10-fold. No AC values < 1 were obtained in any of the subjects. The range of unstimulated activity per g hemoglobin (Hb) was 5 to 12 U. AC values of healthy subjects (1.3) decreased to about 1.15 after vitamin supplementation of 1 RDA for 3 wk. In healthy young subjects consumption of dietary riboflavin at levels as low as 0.5 mg/d resulted in an AC of 1.6.

Effects of dietary pyrazinamide, tryptophan, or nicotinic acid and gamma-ray irradiation on levels of NAD and NADP in various organs of mice.

The effects of large amounts of tryptophan, pyrazinamide, or nicotinic acid in diets on the contents of total NAD (NAD + NADH) and NADP (NADP + NADPH) of various organs were investigated in mice with or without gamma-irradiation. Female C3H/HeN mice were fed one of the following 4 kinds of experimental diets for one week: 1) control diet (20% casein diet containing 3 mg niacin per 100g diet); 2) diet supplemented by 0.5% L-tryptophan (T-diet); 3) diet supplemented by 0.5% L-tryptophan (T-diet); 4) diet supplemented by 0.1% nicotinic acid (NA-diet). Half of the mice in each group were subsequently irradiated with 8 Gy of gamma-ray (60 Co) after 4 h of fasting. Then, the contents of total NAD and NADP in thymus, spleen, kidney, liver, and blood were determined in all animals. The results indicated that NAD content of spleen was higher in PT-group (21.5%) and NA-group (23.2%) than in that of control group. In thymus, however, NAD content of only the PT-group was significantly greater (13.1%) than control. NAD level of kidney was also significantly higher (32.6%) in PT-group. By gamma-irradiation, NAD contents of thymus and spleen of all groups tended to be decreased, but those of kidney and liver were not always reduced. In the latter two organs, significant NAD reduction was shown only in kidney of PT-group and in liver of PT- and NA-groups. Even after irradiation, NAD levels of spleen and thymus in PT- and NA-groups tended to be kept higher than those in irradiated control group.(ABSTRACT TRUNCATED AT 250 WORDS)

The respiratory burst of bovine neutrophils. Role of a b type cytochrome and coenzyme specificity.

A new method of preparation of bovine polymorphonuclear leukocytes (PMN) is described. The subcellular distribution of cytochrome b in resting and activated bovine PMN was compared to that of the O2-.-generating oxidase (assessed as NADPH cytochrome c reductase inhibited by superoxide dismutase). In resting PMN and in PMN activated by phorbol myristate acetate (PMA), cytochrome b was located into two membrane fractions, one of which was enriched in plasma membrane and cosedimented with alkaline phosphatase, while the other consisted of a denser material cosedimenting with markers of the specific and azurophil granules, i.e. the vitamin-B12-binding protein and myeloperoxidase respectively. During activation of PMN by PMA, 15-20% cytochrome b migrated from dense granules to the plasma membrane. The distribution of the O2-. generating oxidase and cytochrome b in subcellular particles was studied during the course of phagocytosis of PMA-coated latex beads by bovine PMN. At the onset of the respiratory burst, the phagocytic vacuoles arising from internalization of the plasma membrane were enriched in oxidase and alkaline phosphatase, but their specific content of cytochrome b was limited; in contrast, cytochrome b was predominant in denser membrane fractions cosedimenting with myeloperoxidase and the vitamin-B12-binding protein. After a few minutes of phagocytosis, a fraction of light vacuoles, slightly denser than the phagocytic vacuoles, became enriched in O2-.-generating oxidase, cytochrome b, the vitamin-B12-binding protein and myeloperoxidase. These vacuoles probably arose from the fusion of the phagocytic vacuoles with dense granules. In bovine PMN supplemented with glucose and maintained in anaerobiosis, activation by PMA induced slow reduction of cytochrome b (60-70% in 15 min at 37 degrees C). Similar results were obtained with cytoplasts after activation by PMA (30% reduction in 3 min at 37 degrees C). Cytochrome b in a particulate fraction obtained by centrifugation at 100 000 X g of an homogenate of PMA-activated PMN, was slowly reduced upon addition of NADPH under anaerobiosis (less 20% in 20 min at 37 degrees C). No reduction occurred in the 100 000 X g fraction prepared from non-activated PMN. The Soret band of cytochrome b reduced by dithionite was displaced by CO only by 1-2 nm. At subsaturating concentrations, CO had no effect on the rate of O2 uptake by activated bovine PMN.(ABSTRACT TRUNCATED AT 400 WORDS)